An infectious bronchitis recombinant virus lacking e protein ion channel activity and its preparation method and application

A bronchitis and ion channel technology, applied in the field of molecular biology, can solve the problems of virion morphological defects, decreased titer, increased apoptosis, etc., and achieves effects of major public health and economic significance

Active Publication Date: 2022-03-22
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In some coronaviruses, the recombinant virus that knocks out the E gene can still maintain the ability to replicate, but its virion form has serious defects, and the titer is significantly reduced. For example, in SARS-CoV, the deletion of the E protein reduces the Pathogenicity and mortality, while recombinant SARS-CoV lacking the E gene led to increased apoptosis and down-regulation of inflammatory factors associated with infected cells

Method used

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  • An infectious bronchitis recombinant virus lacking e protein ion channel activity and its preparation method and application
  • An infectious bronchitis recombinant virus lacking e protein ion channel activity and its preparation method and application
  • An infectious bronchitis recombinant virus lacking e protein ion channel activity and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0096] Example 1 IBV infectious cDNA clone based on seamless cloning technology

[0097] Based on the seamless technology, the E protein gene locus sequence of infectious bronchitis virus was mutated to obtain five fragments spanning the entire IBV genome, namely fragment A, fragment B, fragment C, fragment D and fragment E, Restriction sites were introduced into the 5' and 3' ends of the five fragments, combined with the T7 promoter, ligated in vitro to obtain clones, and transcribed in vitro to obtain full-length RNA (such as figure 1 shown); the specific steps are as follows:

[0098] (1) Infect African green monkey kidney cells (Vero cells, ATCC) with chicken IBV. For the source and infection method of IBV, see the literature "S. Shen, et al. Emergence of a coronavirus infectious bronchitis virus mutant with a truncated 3b gene: functional characterization of the 3b protein inpathogenesis and replication.”.

[0099] (2) The total RNA extracted from IBV-infected African g...

Embodiment 2

[0114] Embodiment 2 The recombination and purification method of rIBV-A26F lacking E protein ion channel activity comprises the following steps:

[0115] (1) First, the E protein gene locus sequence of infectious bronchitis virus is deleted by seamless cloning technology, combined with the T7 promoter, and connected in vitro to obtain a complete RNA clone; then the above clone is infectiously cloned, The cloned sequence to be obtained is introduced into Vero cells by electroporation to obtain infectious bronchitis recombinant virus lacking E protein ion channel activity: the specific operation is the same as

[0116] Example 1.

[0117] (2) The cell supernatant infected with rIBV-A26F was collected, then diluted 10 times with serum-free DMEM, and the virus titer was tested by plaque purification experiment.

[0118] (3) Infect the diluted supernatant virus into a 6-well Vero cell plate, after 2 hours the virus is adsorbed, the supernatant is removed, and the cells are washed ...

Embodiment 3

[0124] Example 3 Stability Analysis of IBV Mutant Attenuated Strain Adapted to Vero Cells Deleting E Protein Ion Channel Activity

[0125] (1) Infect Vero cells with the rIBV-A26F mutant virus, culture at 37°C for 24 hours, collect the supernatant, and then use the supernatant virus to infect newly plated Vero cells, and so on to infect 30 generations;

[0126] (2) Collect, lyse and extract cellular RNA after the 5th, 10th, 15th, 20th, 25th, and 30th generations of virus infection, RT-PCR, E gene sequencing, and compare the sequences to see if there is a new mutation in the E gene, thereby To determine the genetic stability of the strain, the primers of the amplified E gene are the same as those in Table 2, and the sequencing primers are upstream primers.

[0127] (3) Analysis of sequencing results. From the 10th generation onwards, the second T of the 26th amino acid phenylalanine of the rIBV-A26F mutant virus was gradually replaced by C, and the original TTT codon was conver...

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Abstract

The invention discloses a strain of infectious bronchitis recombinant virus lacking E protein ion channel activity, its preparation method and application. The present invention mutates the 26th amino acid of the E protein of the wild-type infectious bronchitis virus from alanine to phenylalanine to obtain the infectious bronchitis recombinant virus lacking the activity of the E protein ion channel. The present invention constructs the infectious bronchitis recombinant virus lacking the activity of the E protein ion channel, and obtains a reversion mutant, and performs separation and purification methods and characteristic analysis of the mutated virus, which is helpful for the development of specific antiviral drugs and An effective vaccine to prevent viral infection has major public health and economic implications for coronavirus research.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a strain of infectious bronchitis recombinant virus lacking E protein ion channel activity, its preparation method and application. Background technique [0002] Chicken infectious bronchitis virus is a coronavirus, first isolated in 1937, that causes significant economic losses to the poultry industry worldwide. The coronavirus is highly contagious and causes respiratory, kidney and fallopian tube disease, weight loss, kidney failure, and decreased egg production and quality. Related studies have shown that coronaviruses can cross species barriers and become pathogens of deadly zoonotic diseases. Currently, there is no effective treatment for coronavirus infection, and the development of attenuated vaccines and specific antiviral drugs is a promising strategy. [0003] The E protein is the key to the pathogenicity of coronaviruses. It is a small protein (76-109 ami...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/01C12N15/50C12N15/63
CPCC12N7/00C12N15/63C07K14/005C12N2770/20021C12N2770/20022
Inventor 冯涛声刘定祥陈瑞爱李淑敏李延鹏梁佳琪袁丽霞朱庆春罗琼熊挺
Owner SOUTH CHINA AGRI UNIV
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